Western blotting is a common lab technique used to detect and analyze proteins. It also happens to be a really long and complicated procedure, with many steps along the way that are easy to mess up. How do you make sure that your Western blot is successful? Avoid the following five ways to destroy your Western blot:
1. Neglect to fill empty lanes with buffer
Unlike an agarose gel, every lane of an SDS-PAGE gel needs to be filled to run properly. Once you’ve loaded all your samples and a molecular weight marker, fill the other wells with comparable volumes of loading buffer. Without this step, your bands will “smile”, turning up noticeably at the ends.
2. Lose track of the front/back, top/bottom; blot the wrong side of the membrane
It’s easy to lose track of where your proteins are on the gel once you’ve begun the transfer. My advice is to develop a rule of thumb that you always follow when removing gels from the gel box. For instance, I always slice a tiny amount of gel off the upper right hand corner of the gel. That way, even if the gel itself gets turned around, you can re-orient yourself.
Within the gel itself, the proteins are evenly distributed. On a membrane, however, the proteins will only be present on one side. Therefore, you also have to keep track of what side of the membrane the proteins are on. To keep the same orientation on the membrane as on your gel, you need to stack them carefully when setting up the transfer – lay the back of the gel on the front of the membrane. If you’re having trouble figuring it out, try to picture exactly how and where the proteins are moving when you apply charge. And don’t forget to mark which side of the membrane is the front when the transfer is complete!
3. Forget to include a ladder
A molecular weight marker needs to be included on every gel! And make sure you use a pre-stained ladder, or you’ll be helpless (ask me how I know this).
4. Get your antibodies wrong
Antibody problems are probably the most common cause for a failed Western blot – using the wrong concentration, using the wrong secondary, or using the wrong reporter. Antibody concentrations need to be optimized for both primary and secondary antibodies; check the literature, the manufacturer’s recommendations, and your lab mates’ experiences for what dilution you should be using. If the antibody is too dilute, you will need a very long exposure to see anything. If it is too concentrated, the signal will be so strong that it will be difficult to distinguish individuals bands. Next, double check that your secondary antibody recognizes the species of your primary antibody, i.e. if your primary is rabbit, the secondary should be anti-rabbit. Finally, make sure your secondary is conjugated to a reporter, such as HRP (hydrogen peroxide), otherwise you’ll just have a stack of antibodies with no way to detect them.
5. Over- or under- expose the blot
When it comes to imaging your blot, determining the exposure length can be somewhat of an art. Under-exposure of your blot will result in very faint bands, and over-exposure can result in an entirely black film. If you always use the same antibody at the same concentration, you probably have a good idea how long the blot needs to be imaged for. It’s a good idea to take a range of exposures, ranging from a few seconds to several minutes.
Well, just as was the case with your agarose gels, breaking your acrylamide gel into many tiny pieces also works really well for destroying your in-progress westerns!
Also, as far as 2D westerns are concerned: any slight slip of the wrist with that thin, tiny, delicate capillary 1st dimension IEF, and that’ll be game over too!
i don’t get why the ladder has to be prestained. i think it’s a good idea for when you’re first working out conditions so you can easily that proteins of all sizes have transferred well, but when you are confident of the conditions, is it really necessary?
also, as far as keeping track of which side of the membran is which, etc., I like to label my blots (with unique ID) in pencil on the top right corner before I do anything else to them — so right on taking them out of thebox, while they’re still dry. a mechanical pencil is the best since the lead is consistently sharp. the indentation from the pencil will transfer all the way through the blotting process so you’ll see it on your film; since it’s not ink, there isn’t anything that leaches out and contaminates.
I’m just guessing, but I would think that the prestained ladder is useful for knowing the correct orientation of your gel when you removed it from your electrophoretic tank. That’s been the case with me- I always hate it when I pre-soak my gel in transfer buffer and then lose track of its orientation.
Thanks for the pencil idea
I like using the prestained markers for monitoring the electroblot… if I accidentally oriented it the wrong way, then at least I know I don’t need to waste my time probing it with my precious primary antibodies, lol. The prestained markers don’t run perfectly though, FYI, so don’t count on them for proper size!
Just a small comment – HRP is not hydrogen peroxide – rather iit s horse radish peeoxidase
You’re absolutely right, Angela – thanks for pointing out my mistake!
“Blocking” can also be tricky. Some antibodies cannot be let in blocking solution, so the incubation has to be done in buffer only, which decreases the quality of the staining afterwards.
Furthermore, incubation with an antibody witout blocking solution should not be performed for too long (overnight)… because then, *surpringly*, no specific signal will be seen!
I’ve not heard of having to fill empty lanes with loading buffer. What is the theory behind this? I did sometimes get smiling even when all lanes are loaded, but changed to pre-casts, which seemed to fix it.
agreed – i dont have smiling without loading all the lanes. that usually happens to me if i try to run it too quickly (high voltage)
Another way to destroy your western blot is by either not washing the membrane for enough time, so as to get fizzy results or washing it for longer times to get no results due to everything being washed off…So keep track of the washing times!!!:)
Another way to destroy a WB: use the wrong buffer for transfer. Preferably one with a high salt concentration so that it boils, burns your membrane, and fuses the whole sandwich together <3